Glycosyltransferases are enzymes involved in in vivo biosynthesis of sugar chains on glycoproteins, glycolipids and the like. Their reaction products, i.e., sugar chains on glycoproteins, glycolipids and the like (hereinafter referred to as “complex carbohydrate sugar chains”) have been shown to be important molecules which play a role in cell-cell and cell-extracellular matrix signaling and serve as tags for complex carbohydrates during differentiation and/or development.
An industrial example where sugar chains are applied is a modification of erythropoietin with sugar chains. Although erythropoietin originally has sugar chains, attempts have been made to increase the number of sugar chains on erythropoietin, whereby erythropoietin products with an extended life span in the body have been developed and are commercially available. In the future, such a product modified with sugar chains is expected to be increasingly marketed. For this reason, production of glycosyltransferases will also be important. Moreover, for functional elucidation of complex carbohydrate sugar chains, various sugar chains are required to be synthesized and their mass production will be necessary.
Synthesis techniques for complex carbohydrate sugar chains are generally divided into two major types. The first is a chemical synthetic approach, and the other is an enzymatic approach using a glycosyltransferase. It should be noted that there is also an intermediate approach, i.e., a chemo-enzymatic synthesis technique based on both chemical and enzymatic approaches.
When a comparison is made between chemical synthetic approach and enzymatic approach, they have both merits and demerits.
As to the merits of the chemical synthetic approach, there are many findings about sugar chain synthesis and hence this approach may be flexibly adapted to the synthesis of various sugar chains. However, the chemical synthetic approach is usually required to comprise a protection/deprotection step and, as a consequence, it involves a long synthetic route and complicated operations. Thus, there is a demerit in that a target product cannot be obtained in high yield. Moreover, as stated above, it is believed that sugar chain modification of proteins, lipids and the like will be important in the future, but it is very difficult to achieve sugar addition without impairing functions of proteins and/or lipids in the chemical synthetic approach in light of its synthesis conditions.
On the other hand, the enzymatic approach is advantageous over the chemical synthetic approach in the following points. In the enzymatic approach, reaction steps are very simple and a target product can be obtained in high reaction yield. Further, since the enzymatic approach allows reactions under mild conditions, it can be adapted to sugar chain modification of proteins or lipids without causing their denaturation.
Until now, about 150 or more glycosyltransferase genes have been isolated from eukaryotic organisms including humans, mice, rats and yeast, and proteins having glycosyltransferase activity have also been expressed in production systems where CHO or E. coli cells are used as host cells. However, enzymes produced by these host cells usually show a very low level of specific activity when compared to the specific activity of glycosyltransferases in their native tissues or cells. This is because although glycosyltransferases produced by E. coli or other host cells have the same primary protein structure as native glycosyltransferases produced in animal cells, there is a difference in the structure or the like added to their protein moiety, so that the specific activity of recombinant enzymes will be reduced when compared to native enzymes.
On the other hand, several glycosyltransferase genes have also been isolated from bacteria which are prokaryotic organisms. Moreover, proteins having glycosyltransferase activity have been expressed in production systems using E. coli and identified for their substrate specificity and/or various enzymatic properties. As to an example of stable and mass producible glycosyltransferases derived from such microorganisms, there are reports of β-galactoside-α2,6-sialyltransferase derived from Photobacterium damselae strain JT0160 (Japanese Patent No. 3062409, JP 10-234364 A). The productivity of this enzyme is 550 U per liter of culture solution, and the enzyme can be presented as an example which is mass producible. However, to achieve more efficient sugar chain synthesis, there has been a demand for the development of a novel enzymatic reaction method for increasing enzyme activity.
For measuring the enzyme activity of mammalian-derived sialyltransferases, it is reported that a divalent ion such as MgCl2 or CaCl2 is often added to their reaction systems (Glycobiology Experimental Protocols, Cell Technology, Supplement, July 1996, pp. 104-107, Shujunsha Co., Ltd., Japan). Also, among proteases extracted from particular types of thermophilic bacteria, some have been known to enhance their activity at a NaCl concentration as high as 1-5 M (Inouye et al. J. Biochem 1997; 122, pp. 358-364).
However, nothing has been elucidated about the effect of NaCl on the activity of glycosyltransferases, regardless of their origin.
Patent Document 1: JP 10-234364 A
Non-patent Document 1: Cell Technology, Supplement, July 1996, pp. 104-107
Non-patent Document 2: J. Biochem 1997; 122, pp. 358-364